PROJECT SUMMARY Membrane fusion, mediated by viral spike glycoproteins, is a key process in the infection cycle of all enveloped human and animal viruses. The overall goal of this project is to understand the molecular mechanisms of Ebola and human immunodeficiency virus entry by membrane fusion, by taking combined state-of-the-art structural and cell biophysical imaging approaches. Although the crystal structures of the ectodomains of several enveloped virus spike glycoproteins have been determined to high resolution and although we and others have contributed NMR structures of the membrane- interactive parts of some of these proteins over the years, we are only beginning to understand the structural transformations that take place when these protein domains interact with membranes and each other, and how these structures drive membrane fusion. In the previous grant period we have made substantial progress towards elucidating the structures of the fusion loop, membrane proximal, and transmembrane domains of the Ebola virus envelope glycoprotein and their pH-dependencies in membrane environments. We have also discovered that HIV particles bind and fuse preferentially at lipid discontinuities in plasma membranes of infected cells and that this process depends critically on the level of membrane cholesterol. Building on these achievements, we now propose to (1) solve the structure of the Ebola virus fusion loop in interaction with its membrane-proximal and transmembrane domain, (2) determine the intracellular factors that trigger Ebola virus fusion in the late endosome, and (3) determine the role of membrane heterogeneity in forming the fusion pore for HIV entry.